Hi
I want to label Gram positive bacteria before exposing RAW 264.7 macrophages to these bacteria (for 1-4 hrs) and then label the macrophages.
Which stain is suitable for the bacterial staining?
Thank
Maya
Do you mean a fluorescent microscopy or flow cytometry method? If so, which wavelength?
There are a couple different ways to go about it. Basically, you would label the cells with an amine-reactive derivative of the dye you want. Traditionally this would be fluorescein, and then use a quenching agent, such as Trypan Blue, in solution with the target cells to quench the labeled bacteria until they are phagocytosed, at which point they become fluorescent.
A more recent technique is to use pHrodo Green or pHrodo Red labeling. These dyes are not fluorescent until they enter a low-pH environment (such as a phagosome), so no quencher is needed and you only get a significant signal upon uptake.
We at Thermo Fisher Scientific sell E. coli and S. Aureus already labeled with these dyes and with traditional dyes, as well as the reactive dye standalone reagents and even kits for labeling bacteria. Consider, as examples, catalog A10026, as an example (pHrodo Red labeling kit for labeling your own bacteria), or A10025 (pHrodo Red labeled E. coli).
You would use the same reactive dye derivatives. The problem, though, is that the reactive dye is optimal at a reaction pH at 8.2. Live bacteria may not be able to withstand that pH. If you lower the pH down to, say, 7.2, the labeling efficiency drops off dramatically and you have to compensate with a much higher concentration of the dye to get the same labeling. So you would first have to determine how high you can go in pH, for the period of the labeling incubation time (maybe 30 minutes?), without killing the bacteria.
Dear Maya Oren Azrad , we use pHrodo kit for phagocytosis assay by flowcytometry and we are very satisfy with the results.
Labelling bacteria with a compatible dye is much of a chemistry thing (as selecting analogue, dye excitation wavelength, emission wavelength, reactivity, and major issue is who will you discriminate the surface adherent bacteria and phagocytosed bacteria). The kit available has the capacity that give fluorescence only if the bacteria is engulfed, not on surface.
Maya Oren Azrad
Hi Maya, do you have the protocol for using bacterial cells for phagocytosis. I am starting out and unsure of how to go about it. Thank you
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